1. Field of the Invnention
This invention relates to and has among its objects the provision of novel methods for the preparation of highly-purified human Antihemophilic Factor preparations. Further objects of the invention will be evident from the following description wherein parts and percentages are by weight unless otherwise specified.
2. Description of the Prior Art
It is known that the clotting of human blood is a complicated process, involving a series of reactions mediated by 13 different factors. It also is well know that a cause of hemophilia is the inability of the afflicted individual to synthesize one of these factors, known variously as antihemophilic factor, AHF, AHG, Factor VIII or Factor VIII C, in amounts sufficient to support adequate clotting. About 40 percent of hemophiliacs have no ability to synthesize AHF, while the others have diminished ability. Dried preparations of AHF concentrate are sold commercially for administration to hemophiliacs for treatment of bleeding or in advance of surgery. The AHF concentrate is obtained from plasma from human donors, through the use of know techniques. At the time of use, the dried concentrate is dissolved in sterile water, and the resulting solution is administered intravenously.
The usual commercial AHF preparation is not pure AHF. Rather, it is an AHF-enriched fraction obtained from plasma and contains other components. It is desirable that the AHF concentrate be as pure as possible, but further improvements in purity through modification of the procedure for isolating AHF from plasma have not been practically feasible due to the difficulty of separating plasma components. AHF is quite difficult to separate and purify because of its low content in the plasma and the instability of its activity. The known AHF concentrates are prepared from fraction I separated from plasma by means of Cohn's ethanol fractionation method or from the cryoprecipitate obtained by freezing a plasma and then thawing it at a low temperature. However, they are all crude products of low purity and contain a large quantity of fibrinogen. If given in large or frequently repeated doses, they may make the state of the patient quite dangerous clinically, bringing an excessively overloaded fibrinogen content in the circulating system. Moreover, it is difficult to determine their accurate dose because of the deviation in the activity in each preparation. For the above-mentioned reasons, the current trend is towards the development of a highly purified, highly concentrated AHF preparation from a large quantity of pooled plasma. The high potency AHF concentrates hitherto disclosed are generally produced by first preparing crude fractions of AHF such as Cohn's fraction I or cryoprecipitate.
The prior art of AHF concentrate fractionation has demonstrated the the concentrate can be separated from fibrinogen and other proteins by column chromatography, polyethylene glycol (PEG) of polypropylene glycol (PPG) precipitation, glycine precipitation (or with other amino acids), as well as alcohol precipitation. British Pat. No. 1,507,198 and U.S. Pat. No. 3,973,002 utilize pH and temperature adjustments to result in some purification of an AHF concentrate to give low potency preparations of intermediate purity. The process described in these patents utilize a fraction extracted from the cryoprecipitate rather than cryoprecipitate itself. Furthermore, fractionation stops after one cycle of pH and cooling adjustment. Others have also recognized the effect of using one cycle of pH and cooling adjustment (J. K. Smith et al, Transfusion 19, 229-306, [1979]).
The compositions of AHF obtained by the processes discussed above are of relatively low concentration (of the order of about 5 to 15 units of AHF activity per ml) and low purity (less than 1 unit AHF activity per mg protein).
Polyethylene glycol fractionation and glycine-precipitated fractionation to yield AHF concentrates have been described in U.S. Pat. Nos. 3,415,804; 3,682,881; 3,770,631; 4,027,013; 3,839,314; 4,069,216; 3,631,018; 3,652,530; 4,073,886; and 4,085,095.
Kisker (Thromb. Diath. Haemorrhagica, 17, 381 [1967]), as well as Penick and Brinkhouse (Amer. J. Med. Sciences, 232, 434 [1956]), have pointed out in their papers, in the preparative process of such high potency AHF concentrates, particularly in the course of separating and purifying AHF the co-existing prothrombin complex and active forms of its constitutive factors, such as IIa, Xa and the like, are markedly detrimental to the stability of AHF and sometimes irreversibly injure the latter to inactivate it. In order to improve stability, yield and solubility of AHF, therefore, it is quite important and essentially necessary to inactivate or eliminate these instabilization factors at the earliest stage of the separation-purification step. Methods for inactivating said instabilization factors have been disclosed in, for example, U.S. Pat. No. 3,803,115. It has also been disclosed that said instabilization factors can be removed by the use of an adsorbent such as aluminum hydroxide, magnesium hydroxide, barium carbonate, barium sulfate, rivanol (6,9-diamino-2-ethoxyacridine lactate), ion-exchange resin (Amberlite IRC-50), glycine ethyl ester or the like (Bidwell, E. et al, Brit. J. Haemat., 13, 568 [1967]; Soulier, J. P. et al, Presse med., 72, 1223 [1964]; Surgenor, P. M. et al, J. Phys. Colloid Chem., 55, 94 [1951]; Hoag, M. S. et al, J. Clin Invest., 39, 554 [1960). Among them, aluminum hydroxide is known to be relatively good adsorbent of said instabilization factors and therefore has been used most frequently (U.S. Pat. No. 4,170,639). DEAE-crosslinked dextran has also been used to adsorb and remove the prothrombin complex (U.S. Pat. No. 4,093,608).
As mentioned above, the AHF concentrates obtained by the prior art processes discussed above are relatively low specific activity, namely about one unit or less of AHF activity per milligram (mg) of protein, one of the undesirable impurities being denatured AHF. U.S. Pat. Nos. 4,289,691 and 4,302,445 disclose processes for preparing AHF preparations having a specific activity of AHF activity of 1-3 units per mg of protein, and U.S. Pat. No. 4,294,826 describes a method for preparing human AHF having a specific activity of about 1-10 units of AHF activity per mg.